The art of optical microscopes is well-established, and a variety of types of microscopes have been developed. In recent years, with progress in peripheral technology starting with laser technology and electronic imaging technology, even more sophisticated microscope systems have been developed.
Against this background, a sophisticated microscope has been proposed that can not only control contrast in the resulting image but that also allows for chemical analysis by illuminating a sample with multiple wavelengths of light to induce a double resonance absorption process (for example, see Patent Literature 1).
This microscope allows for observation of absorption and fluorescence caused by a specific optical transition by selecting a specific molecule using the double resonance absorption. This principle is described with reference to FIGS. 22 to 25. FIG. 22 illustrates an electronic structure of a valence electron orbital of a molecule composing a sample. First, an electron in the valence electron orbital of the molecule at a ground state (S0 state: stable state) illustrated in FIG. 22 is excited by light of a wavelength λ1, and the molecule transitions to a first excited state (S1 state) illustrated in FIG. 23. Next, an electron is similarly excited by light of a different wavelength λ2 and the molecule transitions to a second excited state (S2 state) illustrated in FIG. 24. In this excited state, the molecule emits fluorescence or phosphorescence and returns to the ground state as illustrated in FIG. 25.
Microscopy adopting the double resonance absorption process is for observing absorption images and emission images by using the absorption process in FIG. 24 and the emission of light, such as fluorescence and phosphorescence, in FIG. 25. According to this microscopy, first, the molecule composing the sample is excited to the S1 state by laser light or the like of a resonant wavelength λ1, as illustrated in FIG. 23. At this time, the number of molecules in the S1 state in a unit volume increases as the intensity of emitted light increases.
Here, a linear absorption coefficient is obtained by multiplying an absorption cross-section per molecule by the number of molecules per unit volume. Therefore, in an excitation process as illustrated in FIG. 24, the linear absorption coefficient with respect to the subsequently emitted light of resonant wavelength λ2 depends on the intensity of the initially emitted light of resonant wavelength λ1. That is, the linear absorption coefficient with respect to wavelength λ2 can be controlled by the intensity of the light of wavelength λ1. This indicates that, by illuminating the sample with light of two wavelengths, i.e. wavelength λ1 and wavelength λ2, and taking a transmission image with wavelength λ2, the contrast of the transmission image can be controlled completely by light of wavelength λ1.
Furthermore, if a deexcitation process from the excited state in FIG. 24 to the ground state in FIG. 25 can occur with fluorescence or phosphorescence, the emission intensity thereof is proportional to the number of molecules in the S1 state. Accordingly, it is also possible to control the image contrast when using the microscope as a fluorescence microscope.
Moreover, microscopy adopting the double resonance absorption process allows not only for control of image contrast as described above but also for chemical analysis. That is, since the orbital of the outermost electron in FIG. 22 has an energy level specific to each molecule, wavelength λ1 varies among molecules. Simultaneously, wavelength λ2 is also specific to each molecule.
Even if the sample is irradiated by conventional light of a single wavelength, an absorption image or a fluorescent image of particular molecules can be observed to some degree. Generally, however, since wavelength ranges of absorption bands of several molecules overlap one another, it is not possible to precisely identify a chemical composition of a sample when the sample is irradiated by light of a single wavelength.
By contrast, microscopy adopting the double resonance absorption process uses two wavelengths, λ1 and λ2, to narrow down molecules that absorb or to emit light, thus allowing for more precise identification of the chemical composition of the sample than with conventional methods. In addition, in excitation of the valence electron, only light with a particular electric field vector relative to the molecular axis is intensely absorbed. Hence, taking an absorption image or fluorescence image by determining the polarization direction of the light of wavelength λ1 and wavelength λ2 allows for identification of the orientation direction even for the same molecules.
Recently, a fluorescence microscope that is capable of high spatial resolution exceeding a diffraction limit by adopting the double resonance absorption process has been proposed (for example, see Patent Literature 2).
FIG. 26 illustrates a conceptual diagram of the double resonance absorption process of a molecule. In FIG. 26, the molecule at the ground state S0 is excited by the light of wavelength λ1 to the first excited state S1 and further excited by light of wavelength λ2 to the second excited state S2. Note that FIG. 26 shows that fluorescence from a certain type of molecule in the S2 state is extremely weak.
A molecule with an optical property as shown in FIG. 26 presents a very interesting phenomenon. Like FIG. 26, FIG. 27 is also a conceptual diagram of the double resonance absorption process which shows the vertical axis X representing an expansion of a spatial distance, a spatial area A1 irradiated by light of wavelength λ2, and a spatial area A0 not irradiated by light of wavelength λ2.
In FIG. 27, numerous molecules in the S1 state are generated by excitation with light of wavelength λ2, in the spatial area A0, and at that time, fluorescence from the spatial area A0 emitted by light of wavelength λ3 may be observed. Since the spatial area A1 is irradiated by light of wavelength λ2, however, most of the molecules in the first excited state S1 are immediately excited to a higher state, the second excited state S2, leaving no molecules in the first excited state S1. This phenomenon has been identified for some molecules. Because of this phenomenon, since fluorescence of wavelength λ3 is completely eliminated in the spatial area A1, and since there is no fluorescence from the second excited state S2 to begin with, all fluorescence is completely suppressed (fluorescence suppression effect) in the spatial area A1, with fluorescence only being emitted from the spatial area A0.
Furthermore, when light of wavelength λ2 overlaps a fluorescence emission band, the molecule is forced to transition from the first excited state S1 to a higher vibration level of the ground state S0 by an induced emission process. Hence, the fluorescence suppression effect is further enhanced. In other words, with emission of light of wavelength λ2, a fluorescence yield emitted from the first excited state S1 is reduced. Accordingly, the fluorescence suppression effect is presented if the molecule is forced to transition to a quantum level. Examples of materials having such properties are photochromic molecules, fluorescent substances including rare earth, quantum dots, and the like.
Such a phenomenon is extremely significant from the perspective of the application field of the microscope. That is, conventional scanning microscopes and the like condense a laser beam into a microbeam by using a collective lens and scan the sample. At that time, the size of the microbeam falls to a diffraction limit determined by the numerical aperture of the collective lens and the wavelength. Therefore, further spatial resolution cannot be expected in principle.
In the case illustrated in FIG. 27, however, the fluorescence area is controlled by light of two wavelengths, wavelength λ1 and wavelength λ2, that partially overlap spatially. Therefore, for example examining the emission area of light of wavelength λ1, the fluorescence area can be made narrower than the diffraction limit that is determined by the numerical aperture of the collective lens and the wavelength, substantially allowing for improvement in the spatial resolution. Accordingly, by taking advantage of this principle, it is possible to achieve a super-resolution microscope that adopts the double resonance absorption process and exceeds the diffraction-limited resolution, such as a super-resolution fluorescence microscope.
When using rhodamine 6G, for example, if light with a wavelength of 532 nm (pumping light; first illumination light) is emitted, rhodamine 6G molecules are excited from the ground state S0 to the first excited state S1 and emit fluorescence with a peak at a wavelength of 560 nm. At this time, emitting light with a wavelength 599 nm (erasing light; second illumination light) triggers the double resonance absorption process, and the rhodamine 6G molecules transition to the second excited state, in which fluorescent emission is difficult. In other words, simultaneous irradiation of rhodamine 6G with the pumping light and the erasing light suppresses fluorescence.
FIG. 28 is a configuration diagram of the main section of a conventionally proposed super-resolution microscope. This super-resolution microscope assumes a usual laser scanning type fluorescence microscope and is mainly composed of three independent units, namely a light source unit 210, a scanning unit 230, and a microscope unit 250.
The light source unit 210 has a pumping light source 211 and an erasing light source 212. The pumping light emitted from the pumping light source 211 is incident on a dichroic prism 213 and is reflected thereby. The erasing light emitted from the erasing light source 212 is incident on the dichroic prism 213 after being subjected to spatial modulation of its phase by a phase plate 215, is transmitted through the dichroic prism 213, and then exits after being coaxially combined with the pumping light.
When observing a sample stained with rhodamine 6G, as the pumping light, the pumping light source 211 can be configured, using a Nd:YAG laser, to emit light with a wavelength of 532 nm, which is the second harmonic wave of the laser. As the erasing light, the erasing light source 212 can also be configured, using a Nd:YAG laser and a Raman shifter, to emit light that is the second harmonic wave of the Nd:YAG laser modulated into light with a wavelength of 599 nm by the Raman shifter.
The phase plate 215 modulates the phase of the erasing light and, for example, has a pupil plane radially divided into eight regions about an optical axis as illustrated in FIG. 29. Each region is established by etching a glass substrate or forming optical multilayer films on a glass substrate so that the phase difference of the erasing light revolves about the optical axis over 2π. When the erasing light transmitted through the phase plate 215 is collected, a hollow erasing light with the electric field canceled along the optical axis is generated.
After transmitting the pumping light and the erasing light, coaxially emitted from the light source unit 210, through a half prism 231, the scanning unit 230 performs swing scanning in two dimensions with two galvano mirrors 232 and 233 to emit the light to the microscope unit 250, described below. In addition, with the half prism 231, the scanning unit 230 branches fluorescence that is incident from the microscope unit 250 and travels the opposite path from the outgoing light. The branched fluorescence is received by a photodetector 238, such as a photomultiplier, via a projector lens 234, a pinhole 235, and notch filters 236 and 237.
To simplify the diagram, the galvano mirrors 232 and 233 are illustrated as being swingable in the same plane in FIG. 28. The notch filters 236 and 237 eliminate the pumping light and the erasing light mixed into the fluorescence. In addition, the pinhole 235 is an important optical element composing a confocal optical system and transmits only fluorescence emitted by a particular cross-section in the sample being observed.
The microscope unit 250 is a usual fluorescence microscope which reflects the pumping light and the erasing light incident from the scanning unit 230 with a half prism 251 and collects the light, using a microscope objective lens 252, on a sample S containing molecules with at least three electron states including the ground state. The fluorescence emitted by the sample S is collimated by the microscope objective lens 252 again and reflected by the half prism 251 so as to be returned to the scanning unit 230, while a part of the fluorescence transmitted through the half prism 251 is led to an eyepiece 254 to allow for visual observation as a fluorescence image.
According to this super-resolution microscope, fluorescence is suppressed except near the optical axis, at which the intensity of the erasing light becomes zero on a light collection point of the sample S. As a result, it is possible to measure only fluorescence labeler molecules located in a region narrower than the expansion of the pumping light. Accordingly, by arranging fluorescent signals at each measurement point in a two-dimensional array on a computer, it is possible to form a microscopic image having a resolution exceeding the spatial resolution of the diffraction limit.
In the conventional super-resolution microscope illustrated in FIG. 28, however, during adjustment of imaging performance or microscope assembly for practical use, there is concern that disturbance of the wave front may occur in the erasing light, or that optical adjustment of the erasing light and the pumping light will become difficult.
For example, in the super-resolution microscope in FIG. 28, the phase plate 215 is provided immediately after the erasing light source 212, and after being spatially modulated by the phase plate 215, the erasing light is optically adjusted along the same axis as the pumping light by the dichroic prism 213 and introduced into the scanning unit 230. In this case, optical adjustment of the pumping light and the erasing light becomes particularly problematic. The reason is that if the light paths of the pumping light and erasing light are not aligned to be completely coaxial, the focus spots of these two colors of light do not match on the focal plane.
In other words, in super-resolution microscopy, completely matching the peak position of the pumping light in the central hollow region of the erasing light is a necessary condition. For example, in the focal plane, if the peak position of the pumping light shifts to the periphery of the erasing light, the entire collected pumping light undergoes fluorescence suppression. Therefore, not only does the resolution of the microscope degrade, but also S/N is dramatically reduced.
A super-resolution microscope that can easily align pumping light and erasing light coaxially has also been proposed (for example, see Patent Literature 3). This super-resolution microscope uses the phase plate 300 illustrated in FIGS. 30A and 30B. FIG. 30A is a cross-sectional diagram schematically illustrating the structure of the phase plate 300, and FIG. 30B is a plan view. This phase plate 300 has an annular structure including a central region 310 divided into concentric circular portions and a peripheral region 320. The central region 310 includes optical multilayer films 311 formed on a transparent optical substrate 330 of glass or the like. The peripheral region 320 is, for example, composed of the optical substrate 330. FIG. 31 illustrates optical properties of the phase plate 300. The central region 310 reflects the pumping light and transmits the erasing light while inverting the phase thereof by π. The peripheral region 320 transmits both the pumping light and the erasing light without applying phase modulation.
The phase plate 300 illustrated in FIGS. 30A and 30B can provide the pumping light and the erasing light with different refractive indices by optimizing the total number, film thickness, and material of the optical multilayer films 311. As a result, a phase delay that is an integer multiple of the wavelength can be generated in the pumping light, allowing for the pumping light to be adjusted without undergoing phase modulation. Therefore, the pumping light and the erasing light can be caused to enter coaxially, with only the erasing light formed as a hollow beam.
FIG. 32 illustrates an example of the structure of a super-resolution microscope using the above-described phase plate 300. This super-resolution microscope differs from the super-resolution microscope illustrated in FIG. 28 in the structure of the light source unit 210 and microscope unit 250.
Specifically, the light source unit 210 includes a pumping light source 221, an erasing light source 222, a beam combiner 223 composed of a dichroic prism or a polarizing prism, a fiber collective lens 224, a single mode fiber 225, and the fiber collimator lens 226. The pumping light emitted by the pumping light source 221 and the erasing light emitted by the erasing light source 222 are combined nearly coaxially by the beam combiner 223. The pumping light and erasing light emitted from the beam combiner 223 are incident on the single mode fiber 225 nearly coaxially via the fiber collective lens 224 and are emitted from the single mode fiber 225 as a perfect spherical wave with an aligned solid angle of emission. The pumping light and erasing light emitted from the single mode fiber 225 are converted into a plane wave by the fiber collimator lens 226, which has no chromatic aberration, and are introduced into the scanning unit 230.
When observing a sample stained with rhodamine 6G, a He—Ne laser that emits a continuous wave with an emission line having a wavelength of 543 nm is, for example, used as the pumping light source 221. A He—Ne laser that emits a continuous wave with an emission line having a wavelength of 633 nm is, for example, used as the erasing light source 222.
The microscope unit 250 differs from the structure in FIG. 28 in that the above-described phase plate 300 and an iris (adjustable diaphragm) 261 are disposed along a light path between the half prism 251 and the microscope objective lens 252. The phase plate 300 and the iris 261 are disposed on or near the pupil plane in the microscope unit 250. Only the erasing light is spatially modulated by the phase plate 300 to become hollow. The spatially modulated erasing light and the non-spatially modulated pumping light are then collected on the sample S by the microscope objective lens 252 via the iris 261. Note that the phase plate 300 is optimized for the wavelength of the pumping light and the erasing light. In this way, the sample S is irradiated with erasing light having a beam shape in a hollow pattern with a hollow portion along the optical axis (i.e. a donut shape).
Note that in FIG. 32, the scanning unit 230 and the microscope unit 250 are connected by a pupil projection optical system 270. The remaining structure is similar to FIG. 28, and thus a description thereof is omitted. The super-resolution microscope illustrated in FIG. 32 may be achieved by adding on the phase plate 300 and the iris 261 to a commercial laser scanning type microscope.
Experimental investigation by the inventor, however, revealed that due to defects caused by the principle of the optical multilayer films and the film formation process thereof, the phase plate 300 illustrated in FIGS. 30A and 30B adversely affects the imaging performance of the super-resolution microscope. In other words, by alternately stacking materials that in principle have different refractive indices in the optical multilayer films, interference by multiple reflection of incident light within the films is used to control phase. When the phase plate 215 illustrated in FIG. 29 is configured with optical multilayer films, the glass substrate surface forming the phase plate is divided into multiple regions, and optical multilayer films with different designs are coated so as to generate a different phase delay in each region.
In many cases, an optical design that generates a phase delay functioning as a super-resolution microscope for pumping light and erasing light is possible. Optimization of the corresponding transmittance, however, is difficult. In other words, the beam that has passed through each region varies in intensity in accordance with the transmittance of each region. Therefore, the shape of the collected pumping light and erasing light is disturbed. In particular, since the erasing light is modified to have a hole in the center, axial symmetry is greatly impaired. This leads to degradation of the super-resolution function.
Furthermore, in the optical multilayer films, the refractive index changes suddenly at each layer interface. Therefore, in many cases, even though the refractive index of each layer is extremely low, reflected light is generated at the back face of the layer (back reflected light). In particular, since the erasing light is intense in a super-resolution microscope, erasing light mixes into the fluorescence image as background light due to the back reflected light. Furthermore, in the case of the structure illustrated in FIG. 29, a phase deviated from the design value occurs in each divided region due to error in film thickness during manufacturing. For these reasons, the erasing light has a beam shape that deviates from the theoretical value, resulting in degradation of the super-resolution function.
Currently, two types of phase modulation methods are known as methods for generating hollow erasing light. One method, referred to as a Laguerre-Gaussian beam, changes the phase of the beam around the optical axis by an integer multiple of 2π. Upon collecting such a beam, the electric field intensity cancels out along the optical axis, thus forming a beam with a three-dimensional macaroni shape. In particular, an extremely fine donut pattern is obtained on the focal plane. As a result, extremely high lateral resolution is achieved with super-resolution microscopy. The phase plate 215 with the structure illustrated in FIG. 29, for example, is known as a phase plate used to generate such a beam (also referred to below as a spiral phase plate).
The other method inverts the phase of an annular region in the central portion of the erasing light by π. Upon collecting such a beam, a three-dimensional space that is not irradiated by light is generated only at and near the focal point due to interference of light. In this case, a hollow shape can also be formed in the optical axis direction, and therefore by using this erasing light, the spot contracts in particular in the axial direction, and a super-resolution function in the optical axis direction, i.e. longitudinal resolution, can also be achieved. The phase plate 300 with the structure illustrated in FIGS. 30A and 30B, for example, is known as a phase plate used to generate such a beam (also referred to below as an annular phase plate).
Experimental investigation by the inventor, however, revealed characteristics requiring improvement in both phase modulation methods. Specifically, in a Laguerre-Gaussian beam obtained by using a spiral phase plate, a super-resolution function in the optical axis direction is not obtained since the beam has a macaroni shape. Conversely, a three-dimensional hollow center is obtained when using an annular phase plate, yet since the diameter of the hollow center in the focal plane is larger than the Laguerre-Gaussian beam, a good super-resolution function is not obtained in the lateral direction (see Y. Iketaki, and N. Bokor, Opt. Commun. 285, 3798-3804 (2012)).